In a recent publication, researchers from the University of Miami Miller School of Medicine (USA) describe that Serotonin released by human beta cell inhibits glucagon secretion by alpha cells. They demonstrated that this paracrine loop was mediated via the cAMP pathway. To do so, they captured in live human pancreatic islet cells cAMP signals using a specific fluorescent biosensor.
Screen for inhibitors of PD-1 signalling with a complete cellular assay system
Immunotherapy represents a field in Drug Discovery which is quickly developing and leading to significant progress in treatments of a number of diseases, especially cancer. The approach is based on inducing, enhancing, or surpressing an immune response. Therapeutic manipulation of immunopathways has led to promising clinical results . The first therapeutic antibodies directed against the checkpoint receptor PD-1 have been already brought to the market (Nivolumab, Pembrolizumab) by Bristol Myers Squibb and Merck/MSD respectively, and approved for the treatment of diverse cancer types.
Today, I would like to review tools to build up a comprehensive assay set up for cell based inhibitor screening on PD-1 / PD-L1/PD-L2 binding. [Read more…]
Measure Antibody-Dependent Cell-mediated Cytotoxicity (ADCC)
Fig 1: Antibody-dependent cell-mediated cytotoxicity (ADCC)
ADCC is a simple but important mechanism for the immune system to target diseased or infected cells. Antibodies bind to specific antigens on the surface of the target cell (see Fig 1). PBMCs or natural killer (NK) cells, express Fc receptors on their cell surface and act as the effector cells. Interaction between the Fc region of the antibody and the Fc receptor induces the effector cell to degranulate, releasing IFN-γ, granzymes, and other cytotoxic compounds that lyse the target cell.
ADCC is not only a natural part of the adaptive immune response, but animal experiments have shown that it can also be seen as an important mechanism of action of therapeutic monoclonal antibodies (1), including the breast cancer drug trastuzumab, and rituximab, a drug used to treat diseases which show overactive, dysfunctional, or excessive numbers of B cells (e.g. lymphomas).
Cell lines to build up cellular ADCC screening assays
Fig 2: Principle of BPS’s ADCC cell lines
To enable researchers to build up a cellular ADCC screening system, BPS Biosciences have developed 2 reporter cell lines, which can replace NK cells or PBMCs in such a cellular assay (see Fig 2). The system is based on Jurkat cells that stably express human FcγRIIIa (CD16a), the receptor for the Fc region of human IgG. The FcγRIIIa on the Jurkat cells binds to the IgG on the surface of the target cell. This crosslinking causes the Jurkat cells to activate NFAT transcription, which induces the expression of luciferase and can be easily detected using the ONE-Step™ Luciferase Detection Reagents.
The effectiveness of ADCC depends on how well the effector cells are activated after the engagement of FcγRIIIa. Human FcγRIIIa displays dimorphism at amino acid 158 – one allele (V158) encodes a high Fc affinity receptor variant, while the other (F158) encodes a lower Fc affinity receptor variant. BPS offers 2 different ADCC cell lines expressing either of these Fc receptors to allow selective antibody binding analyses using each type of receptor.
- ADCC Bioassay Effector Cell, F variant (Low Affinity)
- ADCC Bioassay Effector Cell V variant (High Affinity)
Get more information about our ADCC cell lines – just leave your questions or comments in the form below!
Reference:
(1) Clynes, RA, Towers, TL, Presta, LG, Ravetch, JV; Inhibitory Fc receptors modulate in vivo cytotoxicity against tumor targets; Nat Med. 6 (4): 443-446 (2000)
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Cancer Immunotherapy: Targeting CD73 to inhibit adenosine generation
Fig 1: CD39 and CD73 leading to elevated levels of extracellular adenosine.
Extracellular adenosine 5′-triphosphate (ATP) is released by dying and damaged cells, and it acts on many immune cells to promote inflammation. On the other hand, the unphosphorylated
metabolite, adenosine, functions as an anti-inflammatory molecule. Two extracellular ecto-5´-Nucleotidases, CD39, and CD73, convert extracellular ATP to ADP/AMP and AMP to adenosine, respectively, leading to elevated levels of extracellular adenosine (Fig 1). [Read more…]
New research tools targeting PCSK9 and LDLR
There is an increasing demand for new cholesterol-lowering therapies in addition to existing treatments (e.g. targeting statins). The pharmacological inhibition of PCSK9-mediated LDLR degradation is becoming very attractive for treating cholesterol-related diseases. But which reliable in vitro assays are available to effectively study “PCSK9-LDLR” interactions ?
Cancer Immunotherapy – Investigate the TIGIT/CD226 Pathway
Fig 1: TIGIT (VSTM3)/CD226 and CD28/CTLA-4 Pathway
Over the past months, we have released a number of posts introducing inhibitor screening assays intended to investigate immuno checkpoint protein interactions, such as PD-1/PD-L1/PD-L2; B7-1/CD28, B7-1/CTLA4; BLTA/HVEM, CD47/SIRPα; GITR/GITRL and many others – for an overview, you might like to read 9 pathway-specific screening assays in Immunotherapy.
Today, I’d like to concentrate on the TIGIT/CD226 pathway, because it acts through a novel mechanism to regulate CD8+ T cell functions within the tumour microenvironment. [Read more…]
New Polyethylene Glycols (PEG) as versatile biochemical linkers
Polyethylene Glycols (aka. PEGs) are biologically inert, non-immunogenic, and hydrophilic molecules. These properties make them very attractive for research and drug discovery applications (ex. Antibody-Drug Conjugation, imaging, enzymatic detection). The aim of this post is to introduce recent developments in the field of PEG technology provided by BroadPharm.
Best methods for Circulation miRNA profiling (cmiRNA)
We need to find biomarkers for prognostic, diagnostic and personalised treatment development. Notably to fight cancers that affect tissues. Since biopsies are invasive, it’s better to look for biomarkers in body fluids. Indeed, a simple blood sample becomes a kind of ‘liquid biopsy’ to reveal tissues affections. For 13 years, increasing interest has been shown for miRNA as biomarkers and it will last for sure. The 2 main reasons are that they are major regulators of cell processes and they are released from tissues into the blood. They are major biomarker candidates in serum and plasma. Thus, these circulating miRNA (cmiRNA) are the best hope for modern medicine. Still, a lot of research has to be done to determine the specific signature for each pathology, and also depending on the patient background. Obviously, cmiRNA profiling is a key step and requires sensitive and reproducible method. Sequencing, qRT-PCR, several kind of microarrays… Let’s explore together what the best approach could be. [Read more…]
Designer Nucleosomes – the next generation of epigenetic substrates!
Fig. 1: Mononucleosome structure
In eukaryotic cells DNA is packaged in nucleosome units called mono-nucleosomes which consist of a segment of DNA called core DNA (147 bp in length) wound around a histone octamer (Fig 1.). Histone octamers are assembled from 2 copies of the the core histones H2A, H2B, H3, and H4. These mono-nucleosomes are connected by linker 80bp-long DNA. A fifth histone type of protein, the so-called linker histone H1, binds to the linker DNA close to the entry and exit of the core DNA and is involved in chromatin compaction (Fig 1.).
Substrates for epigenetic enzyme assays and inhibitor screenings
In the past, native or recombinant nucleosomes, single histone proteins, or histone derived peptides have been available for assays with epigenetic enzymes such as histone methyltransferases or histone acetylases and related screenings for inhibitors of the respective enzymes (for an overview about the products available, you might like to read my recent post Find the best epigenetic enzyme substrate for your needs).
Table 1: Specific histone modifications can be linked to certain diseases and effector proteins (provided by Epicypher).
As some epigenetic enzymes require highly specific substrates, Epicypher has now launched a new product line – Designer Nucleosomes (dNuc). dNUCs are semi-synthetic nucleosomes incorporating specific histone post-translational modifications. These reagents represent a powerful new technology – critical in understanding chromatin biology and for the development of novel drug targets and precision therapeutics. It is known that highly specific histone modifications can be linked to certain diseases (see Table 1). Table 1 liste the dNUCs which are already available, for further information have a look at our list of Designer Nucleosomes. dNUCs serving as substrates for the most relevant epigenetic enzymes will be added to our catalog in the coming months. If you have specific modifications in mind, which which are know to be optimal for your enzyme of interest, please let me know through the form below. Epicypher might already have this designer nucleosome in their pipeline – or might be able to produce it on a customized basis.
To get more insight into the dNuc manufacturing process Epicypher applies, you can download the white paper Not All Designer Nucleosomes are Created Equal: A Tale of Two Cysteines.
The paper compares the two currently used synthetic methods to produce dNUCs, native chemical ligation (NCL) and methyl lysine analog (MLA). The paper shows that NCL – the method used by Epicypher – yields superior nucleosome preparations.
Any questions or comments? Please feel free to contact me with the form below!
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IL-17A pathway – a key target for immunotherapies
Fig 1: Function of IL-17 in diverse processes
Interleukin-17 (IL-17) is a family of 6 closely related cytokines, designated IL-17A-F, that play a central role in mediating inflammation, autoimmunity, and host defense. IL-17 is mainly secreted by a specific subset of T helper cells known as TH17 cells (for an overview see Fig 1). The IL-17 cytokines mediate their biological functions via surface receptors on target cells. IL-17A binds to IL-17 receptor A (IL-17RA), which stimulates the production of other pro-inflammatory cytokines including IL-6 and IL-8. IL-17A and its receptor play a pathogenic role in many inflammatory and autoimmune diseases such as rheumatoid arthritis. IL-17 signaling is also involved in mucosal immunity and host defense against extracellular bacterial and fungal infections (Staph, Candida, Pneumonia, etc.). [Read more…]
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